Vegetable



United States Patent PROCESS OF ISOLATING HYPOTENSIVE AGENT FROMVERATRUM PLANTS Gerhard W. Kusserow, Santa Monica, Murle W. Klohs,Glendale, and Philip K. Bates and Kenneth J. Gross, Santa Monica,Calif., assignors to Riker Laboratories,

Inc., Los Angeles, Calif., a corporation of Delaware Application April6, 1950, Serial No. 154,324

13 Claims. (Cl. 26l )236) This invention relates to hypotensivecompositions and to the preparation of hypotensive compositions; moreparticularly it relates to the extractives and to the extraction of suchhypotensive alkaloidal compositions from the drug Veratrzmz viria'e,also known as American hellebore and green hellebore, and from others ofthe Veratrum genus, such as Veratrum album, Verajrum californi cu m,Veratrum fimbriqtum, and Veratrum eschscholtzz'i, and from similar plantdrugs containing hypotensive alkaloids.

This application is a continuation-in-part of our application Serial No.92,256, filed May 9, 1949, now abandoned.

While many compositions have been developed and used to lower bloodpressure, and the search for more satisfactory remedies for circulatoryailments, including hypertension in particular, has been active, thosecompositions so far placed at the disposal of the medical professionhave been characterized by many disagreeable side reactions, or havebeen so difilcult and hence ex: pensive to prepare that their usefulnessin the hands of the physician has been greatly impaired.

We have discovered that hypotensive alkaloidal compositions ofoutstanding therapeutic value may be obtained from various species ofthe genusVeratrum and kindred drugs, more particularly Veralrum viride,Verarur n escholrzii, and Veratrum fimbriazum, and we have alsodiscovered novel and efiective processes for readily segregating suchhypotensive compositions.

The compositions derived by our process from Vera- 2mm virz'de andsimilar species are alkaloidal and have definite activity in loweringblood pressure when administered orally, and have definitephysico-chemical characteristics when subjected to the standard testsfor purity of organic compounds. Because of their definite therapeuticactivity, the physician is enabled to determine readily and speedily theproper dosage for the individual patient. Further, our compositions arenonhabit forming and are especially valuable in that optimum dosages maybe administered orally over a considerable period of time withoutuntoward results, building up of tolerances or disagreeable sidereactions.

7 One object of the invention, as indicated in the foregoing, is toprovide the medical profession with an improved hypotensive agentderived from Veratrum species such as vixide.

Another object of the invention is to provide a process for theextraction of hypotensive alkaloids from the dried rhizome and roots ofVeratrum viride and other species.

Another object of the invention is to isolate in a substantially pureform the desirable alkaloidal components from the extractives of thedried rhizome and roots of Veratrum viride and other Veratrum andsimilar alkaloidal drug plants.

Another object of the invention is to isolate in the form of analkaloidal composition those alkaloids derivable from Veratrum speciesand similar alkaloidbearing plants which are useful and desirable asbloodpressure-lowering agents, or hypotensive agents, while eliminatingalmost completely all of those accompanying alkaloids which producetherapeutically objectionable side reactions. We have found that many ofthe better known .Veratrum alkaloids are not importantly valuable ashypotensive agents and in addition produce objectionable side reactions.They include jervine, rubijervine, isorubijervine, pseudojervine,veratramine, veratrosine, and the like, and these we seek to eliminate.

Other objects will become apparent from the following specification, inwhich Veratrum viride is taken as typical of the various Veratrumspecies and similar drug plants containing alkaloidal hypotensiveagents.

We have found that a menstruum comprising a solvent for the alkaloidalmaterials, such as chloroform to which aqueous ammonia (ammoniumhydroxide) has been added, extracts the alkaloidal compoundssubstantially completely from dried ground rhizome and roots of Vera:trum viride. C hloroform substitutes may be employed in some instancesas hereinafter indicated, and, especially, instead of chloroform withaqueous ammonia, anhydrous chloroform or equivalent saturated withammonia gas may be substituted. The resultant extracted substance, whichis a resin-like material, contains not only the de.

- sired alkaloids but also impurities and other alkaloidal constituentswhich produce objectionable side reactions when used therapeutically.Without at present committing ourselves to any particular theory, whileit is possible that the ammonia (aqueous ammonium hydroxide) merelyincreases the solvent efiect of the chloroform, it now seems moreprobable that the desired alkaloidal constituents are naturally presentinthe drug plant as acidic salts and that these are converted by ammoniato free, alkaloid bases which are soluble in chloroform (or similarsolvent). 7

When fresh or undried rhizome and roots are to be treated, owing totheir Wet condition they are initially extracted with aqueous aceticacid, precipitated with ammonia, and then extracted with ammoniacalchloroform or the like as more fully developed later. This acetic acidextract from the fresh or undried materials is similar in alkaloidalcontent to the resin-like material obtained by starting with chloroformand ammonia. It is probable that the acetic acid (or equivalent acidherein disclosed) converts the natural alkaloid salts in the drug plantto water-soluble salts which are readily soluble in the water of theacid solution.

An important aspect of this invention is the isolation from theabove-described extract of the desirable alkaloid constituents whichlower blood pressure without produc ing therapeutically undesirable sidereactions when administered to the patient, the objectionable, morecommonly known alkaloids of the various Veratrum species and variousimpurities being substantially or entirely eliminated. Thus, a pluralityof desired alkaloids is recovered as the new alkaloidal composition.This composition is made up of amorphous bases not at present positivelyidentified and perhaps one or more of the less known alkaloids of thegroup commonly derived from the various Veratrum species.

In the accompanying drawing, the figure is a flow diagram indicative ofthe general steps of the process of isolation and purification, and alsoindicative of one acceptable procedure, although variations thereof aredisclosed in the following specification.

As a first step in one method of purification, the solvent is removedfrom the resin-like extract as by evaporation under partial vacuum at aslightly elevated temperature, for example, 30 C. to 50 C., whichinsures against decomposition of the alkaloids. The solvent-free extractis then dissolved in one of the lower alcohols, such as methanol,previously slightly heated.

results are obtained. A few per cent, such as 4% or 5%, constitute apractical limit. As a matter of fact, if the percentage is carried toohigh, there may be a tendency to reduce the proportion of alkaloidsextracted from the plants. If a solvent such as methanol is employed,where it is possible to absorb as much as 20% of ammonia gas, such anammonia gas concentration is to be avoided both because of the tendencyto lower extraction capacity, and to avoid saponification which mayoccur if the plant drug contains any significant proportion of moisture,as when the plant drug is damp or wet.

Purification of crude extract The resinous chloroform extract isdissolved in about 440 ml. of warm methanol (about 50 C.) (2 ml. pergram of extract). The methanol solution is then poured into about 4.4liters of 5% aqueous acetic acid, with stirring. This is about onevolume of methanol to about volumes of acid solution. After standing forone hour the solution is filtered through ordinary towel cloth, thenthrough a polishing filter using an asbestos filter disk. The clearorange brown filtrate is adjusted to a pH of about 9.2 with concentratedammonium hydroxide diluted with an equal volume of water to yield about14% ammonia by weight. A cream-colored precipitate forms and isrecovered by filtration. After being thoroughly washed with water theprecipitate is dried in a vacuum oven at about 35 C. for about 15 hourswith some flow of air through the oven. When dry, the material is acream-colored powder which will be referred to as the water-insolublefraction containing the desired alkaloids. The yield is about 0.35% to0.40% (Product A).

The intermediate cream-colored product (Product A) may be used as suchwithout further purification because it possesses valuableblood-pressure-lowering properties without significantly objectionablefactors. its physicochemical characteristics are as follows:

Descripti0n.An off-white, cream-colored powder which is verysternutative;

Solubility.Easily soluble in many organic solvents such as methylalcohol, ethyl alcohol, chloroform, acetone, propylene glycol, ethyleneglycol monomethyl ether (methyl Cellosolve); partially soluble inbenzene and ether (ethyl ether); substantially insoluble in water;

Optical rotation range.[al =73 to ll0 at a concentration of 0.5 gram per100 ml. in 95% ethyl alcohol;

Ultraviolet absorption-Strong absorption peak at 250 millimicrons atwhich the calculated specific extinction coeflicient is approximately19,000 to 23,000 based on a concentration of 0.000016 gram per ml. of95% ethyl alcohol;

Nitrogen c0ntent.--Approximately 2.9%;

Sulfuric acid color test.When dissolved in concentrated sulfuric acid ona spot plate, gives a yellow color which changes to brown on standing.

As previously indicated, this product, in addition to the statedsolubilities above, is also soluble in dioxane, in acetic acid and otherdescribed acids, and in ammoniacal chloroform, but is insoluble inammonium hydroxide solutions and similar aqueous basic solutions, atleast at a pH above about 8.

Separation of the desirable alkaloids from the waterinsoluble fractionThe water-insoluble fraction obtained above (approximately 52 gms.) isdissolved with stirring in about 1000 ml. of 5% aqueous acetic acid(about ml. per gram of the precipitate constituting the above ProductA). To this solution is added about 100 to 105 gms. of reagent gradeammonium sulfate dissolved in 250 ml. of water (about solution). Afterstanding for one hour the resultant precipitate is removed by filtrationand washed with water on the filter. The filtrate and washings arecombined and adjusted to a pH of 9.4 to 9.5 with concentrated ammoniumhydroxide and the precipitate formed is recovered by filtration. Aftermost of the Water has been removed from the gelatinous precipitate onthe filter funnel, the precipitate is dissolved in an excess ofchloroform by shaking in a separatory funnel. The chloroform layer isremoved and dried with anhydrous sodium sulfate. The dried chloroformlayer is evaporated to dryness under vacuum and the residue dissolved asmuch as possible by shaking with 250 ml. of benzene (benzol). Thebenzene solution is filtered and the filtrate evaporated to drynessunder vacuum, yielding a pale yellow product that contains the alkaloidsmost desirable for blood pressure lowering. The yield is about 0.06% to0.10% (Product B).

This pale yellow product is preferably further treated to remove theremaining less desirable rubijervine and isorubijervine constituents byshaking with about 250 ml. of acetone to dissolve the constituentssoluble therein, filtering and recovering the solution, and evaporatingto dryness under vacuum to yield a final pale product which may bedesignated as straw-colored. The yield is about .04% to 07% (Product C).

Products B and C, as apparent from the foregoing descriptive matter, arewater-insoluble, soluble in solutions of nonoxidizing and nonreducingacids of the character indicated, soluble in the lower alcohols, solublein dioxane, and soluble in chloroform and ammoniacal chloroform, butinsoluble in aqueous alkaline solutions such as ammonium and alkalimetal hydroxide solutions, at least above a pH of about 8. They are alsosoluble in benzol, and acetone, and moreover are soluble in indicatedaqueous acid solutions containing ammonium sulfate, whereas some of theobjectionable alkaloids which are soluble in the acid solutionsthemselves are precipitated as sulfates in the presence of ammoniumsulfate. Similarly, while the desirable alkaloids are precipitated fromsuch an acid solution, containing ammonium sulfate, upon addition ofammonium hydroxide to render the solution alkaline, certain undesirablealkaloids remain in the solution and are discarded upon filtration.

The processes herein described are desirably carried on in glass orsimilar apparatus, or in stainless steel apparatus, to insure againstpossible contamination by materials of which the apparatus mightotherwise be constructed.

Except as otherwise indicated as important and limiting, operations areordinarily conducted under room temperatures and pressures, andproportions may be varied considerably as will be understood by thechemist. In connection with drying temperatures, where reference hasbeen made to drying at about 30 C. to 40 C., it will be appreciated thatboth higher and lower temperatures may be employed, according to theparticular drying method used. if higher temperatures are employed, theyshould not exceed about C. because decompositlon starts at about C. andbecomes serious at about C. Similarly, considerably lower temperaturesmay be employed where low temperature drying processes are acceptable,and possibly drying may then occur at temperatures as low as 70 C. inconnection with employment of warm solvents, the upper temperaturesordinarily will be readily controlled by reason of the comparatively lowboiling point of the solvent. Otherwise, it is obvious that thetemperatures will be regulated to avoid decomposition.

Obviously, many modifications of the process as above described, may bemade without departing from the spirit of the generic inventionpresented. Thus, acetone, benzene, ether, or other volatile organicalkaloid solvent capable of absorbing ammonia and inert or nonreactivetoward the alkaloids, such as carbon tetrachloride and the like, may besubstituted for chloroform in the initial extraction of the groundVeratrum viria'e or other Veratrum species. Other lower aliphaticalcohols up to about eight carbons per molecule, such as ethyl, propyl,isociablyihigher thanpH 7.

propyl, amyl, and hexyl alcohols and mixtures thereof, or oth erWater-.mis cibIesQIVents or :mixtures thereof, maybe Substituted formethanol in the process. Such water miscible solvents are particularlythe water-miscible others, suchas m-dioxane and p-dioxane,-and the alkylethers of diethylene glycol (commonly known as the fCarbitols) suchasthe monomethyl,nronobutyhmonoethyl, and diethyl'diethylene glycols.Other examples of such water+miscible solvents are .glycerine, propyleneglycol and the methyl, ethyl, butyl, and benzyl glycol ethers known asthe Cellosolvesl Instead of the acetic acid particularly mentioned,other dilute acids; maybe used, forexample, citric acid, tartaric acid,sulfuric acid, vhydrochloric acids and mixtures thereof. In practicethese acids are commonly used in relatively=di1ute watersolutions,'su'ch as the 5% acetic acid solution previously mentioned. ineach instance Where acid generically is mentioned, it is understoodthat, 'for'tliis purpose, the acid shall be a nonoxidizing, nonreducing,dilute, aqueous acid solution, the acid being a freely water-solubleorganic or inorganic acid capable of combining with the alkaloidalconstituents present to form the water-solubleacid addition salts of thedesirable alkaloidsas well understood in this art. By this definition itis intended to exclude such acids as nitric acid, sulfurous acid,chromic acid, perchloric acid, and thellike, 'at least under conditionsin which oxidization or'reduction might takeplace. This, of course, willbe obvious to the organic chemist.

Y-Similarly, where the use of ammonium hydroxide has been indicated, thealkali metal hydroxides maybe employed to replace ammonium hydroxideexcept Where the solventemployed must be volatile as in the case oftheinitial chloroform extraction step in which ammonium hydroxideisused. Otherwise mixtures of these alkaline materials,.includingammonium, sodium, and potassium hydroxides andmixtures thereof, orotherappropriate alkaline.solutions,.may be used interchangeably. Commonlythese alkaline materials are used in concentrations -to yield a pH valueof about 9.2, and sometimes a;pH value of 9.4 or 9.5.. The usualoperative range is between about pH8 and vpH 10, or in other-Words,appre- Ordinarily a pH above 10 will not be employed because a higher pHtends .to saponification or kindred reaction with the desiredalkaloidal:materials undergoing treatment.

In connection with the previouslymentioned use of anhydrous dryingagents, in addition to employment of anhydrous sodium sulfate, otheranhydrous drying agents,'such as calcium sulfate, calcium chloride, and

copper-sulfate, may be employed, as may .many of the well-knownmeans forremoving fluids from precipitates or separating immiscible liquids, suchas centrifuging, whichyieldsatisfactory-results when employed in theprocessv insteadof filtering.

Respecting the employmentofammonium sulfate to precipitate undesiredalkaloids, the proportion used is between aboutone-quartergram andabout. four grams per, gram of dissolved precipitate in the solutionbeing treated. The optimum is about two grams ofammonium sulfatqper gramof dissolved precipitate. The proportion of about four grams isapproximately the .upper practical limitbecause, as the proportion offour grams is exceeded, such greater proportion of ammonium sulfatebeginstothrow out the desired alkaloids. It will be noted thattheammoniurn sulfate solution indicated in theabove example is a fairlystrong solution containing about of; ammonium sulfate. Such strongsolution is desirable inasmuch. as it reduces the volume of totalsolutionnto be handled. Instead of ammonium sulfate it issometirnesfeasible to use suchzsulfates as the alkali metal sulfates and thealkaline earth metal sulfates. Since. it isthe small concentrationof thesulfate. radical which important, it will sometimes be possible, thoughapparently not so desirable, to use a minor proportiou of sulfuric acidfor :example around 1% to 1.5% 11-1280. based on thetotal-solution. I g4 in addition to the pale yellow productobtained as abovedescribed,which contains the most desirable alkaloids, other alkaloidsmay beobtained'from'the ammoniated filtrates indicated .in the purificationand separation paragraphs of the above example, appropriate solvents andprecipitants, such asthose herein disclosed, being-employed to separatethe respective alkaloids :by steps similar to those steps describedabove.

Having particular reference to the pale yellow product (Product B) ofthe example, this blood-pressurelowering alkaloidal fraction possessesthe following physico-che'rnical characteristics Sdlubility.Easilysoluble in most'organic solvents such as methyl alcohol,.ethyl alcohol,chloroform, ben-' zene (benzcl), ether, acetone, ethylene glycolmonomethyl ether (methyl Cellosolye), ethyl acetate, methyl ethyl ketoneand propylene glycolysubstantially.insoluble in water, the lower boilingpetroleum fractions such-as petroleum ether, and cyclic paraflins;

Optical rotation range..-[a] =l5 to 35 ata concentration of 0.5 gramper100 ml. in 95% ethyl alcohol;

Ultraviolet adsorption-.Absorption,peak at 250 .to 254-millimicronsat-Which thecalculated specific extinctioncoeificient isv approximately4,000 to 6,000basedon a concentration 01' 000008 .gram permleof 95%ethyl alcohol; there is also a definite leveling oifin the absorptionspectrum curve at 280 to 3 00 millimicronsand the calculated specificextinction coefiicient using 290 millimicrons as an average isapproximately 820 to .1640 based on a concentration of 0.0004 gramgperml. of 95% ethyl alcohol; I v g Nitrogen content. Approximately 2.9%;

Sulfuric acid color rest.-When.dissolved ,in concentrated sulfuric acidon aspot plate, gives a bright orange brown colorwhich changes to alightbrown upon-stand- Having reference to the straw-colored .finalzproduct(Product C) of the example produced by a. final-extraction withacetone,the characteristics of thiscomposition are in general the same as thoseabove given for the; pale yellow product, except as differentlyindicated below. These characteristics areasfollows:

.Color, appearance, taste.-Straw-colored ,powder, strongly sternutatory,and having a bitter acrid taste;

Optical 1 r0tazion.['c] ='-25 :7 (Concentration =0.5% in ethanol);

Spectrophotomezric characteristics.-

Log E250 ma 3.75 0.20 Log E290 mu=3.02i0.20 (Unit of concentration 1gm./cc. in'ethanol);

Equivalent weight.550i50;

Nitrogen based an equivalent weight.--2.33% :10 2.80%; v

Percent v0latilematter.Not greater than 5% when determined at 1 mm. Hgat 70 C.;

Melting p0int.135 C.;

S0lubility.Distilled watch-Insoluble. 99%+ soluble at a concentration of40 mg. per cc. of solvent at'25C. Benzene.%+ soluble at a concentrationof 80 mg.'per cc. of solvent at-25 C.

Having reference to the .previouslyindicated acetic acid extraction offresh or undriedVeratr imyiridegland similar plants containinghypotensive a gcnts,.fth e extraction is made with dilute acetic acid.(or dilute. equivalent acid asheretofore disclosed). Uponcompletionofthe, acid extractiomthe solution is removed. madetarni moniacal [(e. -g.pH 9.2) with ammoniumfhydroxide, thereby producing aprecipitate .which.is yrecovereld z by, filtration. Theiiltrate. maybe treated for recoveryofita product as hereinafter disclosed. The precipitate is taken Acetoneup in ammoniacal chloroform produced as heretofore described, orequivalent solvent, the undissolved residue being discarded and thefiltrate being evaporated to dryness under vacuum to yield a solidproduct. (This procedure may also be used on the dried plant drugs, butis not economically desirable for that purpose.) This productcorresponds with the cream-colored intermediate product above described(Product A) which is precipitated from an acetic acid-alcohol solutionor from an acetic acid-dioxane solution by rendering alkaline withammonium hydroxide, and then recovered as a precipitate. Thisacid-extracted residual product obtained by the evaporation of theammoniacal chloroform may be used as such, or mixed with the describedintermediate cream-colored product. Also, it may be further purifiedwith, or in the same manner as, such intermecfiate product to yieldfinal products corresponding with those pre viously described (ProductsB and C). Like the creamcolored intermediate product, this residualproduct recovered by the evaporation of the ammonia-chloroform solventis substantially alkali-insoluble, water-insoluble, soluble in thedescribed acids, soluble in chloroform, and soluble in the describedalcohols or others such as methanol or dioxane.

EXAMPLE As an example of a specific procedure of acid extraction of afreshly harvested (undried) plant drug, the following is given:

Extraction of plant Approximately 8 pounds of ground, undried, greenhellebore containing about 85% moisture is placed in a glass percolatorand covered with 10% aqueous acetic acid (about 8 pounds). Afterstanding for 16 hours the extract is drained.

Nora-Taking into consideration the moisture content of the drug in thisexample the acetic acid in the total water present is about 5.5% and theratio of total menstruurn to dry material (solids) is in the order of 12to 1 by weight. These figures are not critical. The acid strength canvary from 5% to 6% and the ratio of menstruum to solids from, say, 10 to1 up to 14 to 1. However, the concentration and quantity of menstruumadded should preferably be adjusted within these limits based on theapproximate moisture content of the drug.

Purification of crude extract The aqueous acid extract is adjusted to apH of 9.5 with concentrated aqueous ammonium hydroxide (28% NHa). Theprecipitate formed is recovered by filtration, and after beingthoroughly washed with water is dried in a vacuum oven at about C. withsome flow of air through the oven. The yield is 0.3% to 0.5% (dry basisfor starting drug).

The dried material is then treated with about 300 ml. of chloroform towhich 10 ml. of 28% aqueous ammonia has been added (or preferably with achloroform solution of gaseous NHa) to dissolve as much as possible ofthe material. The chloroform-insoluble residue (which is chiefly inertimpurities) is removed by filtration and the chloroform extract isevaporated to dryness under vacuum. The material, when dry, is acream-colored powder corresponding with that heretofore referred to(Product A) as the water-insoluble fraction containing the desiredalkaloids and also designated as the intermediate product. The yield is0.15% to 0.25% (dry basis).

As previously indicated, other useful alkaloids may be obtained from theammonia filtrates obtained upon precipitation with ammonium hydroxidefrom acetic acidalcohol solutions (or other acid-alcohol solutions ofdisclosed acids and alcohols), or from acetic acid-dioxane solutions (orother acid and water-miscible ether or similar solutions disclosed).Similar desirable alkaloids are similarly obtainable from the abovedescribed ammonia filtrate obtained upon neutralization with ammoniumhydroxide of the acetic acid solution produced in extracting fresh orundried starting materials. Both types of ammonia filtrates mentionedare to be treated in the same manner, and may be combined for suchtreatment. The treatment consists in extracting desired constituentsfrom the ammonia filtrates with chloroform. Chloroform is admixed in anypreferred manner with the aqueous ammoniacal filtrate, as by agitationtherewith in sufiicient quantity to extract from the aqueous ammoniafiltrate all of the chloroform-soluble constituents. The chloroformsolution is then allowed to stratify and separate from the aqueouslayer.

The chloroform solution is then evaporated to dryness under vacuum toyield a chloroform-soluble residue which constitutes a therapeuticallyuseful intermediate product. This may be designated as Product D.

A further purified product may be obtained from the chloroform-solublefraction by ether extraction, any undissolved residue being discardedand the ether solution evaporated to dryness under vacuum to yield thedesired further purified product. This may be designated as Product E.

More specifically, the clear, aqueous ammoniacal filtrate used as thestarting material for this phase of the invention, which commonly has apH of about 9.2, is extracted in a continuous extractor with chloroformfor about sixteen hours or more until all of the chloroformsolubleconstituents have been taken up in the chloroform. This chloroformextract is then evaporated to dryness under vacuum. It is partiallywater-soluble. It contains desirable hypotensively active alkaloids. Theyield is about 0.09% to 0.14%. The physicochemical characteristics ofthis chloroform-soluble extract (Product D) are as follows:

Solubility-Readily soluble in methyl alcohol, ethyl alcohol, chi reform,propylene glycol and methylCellosolve; partially soluble in dioxane,acetone, ether, benzol and water;

Optical rotation range.-lul =l0 to 25 at a concentration of 0.5 gram per100 ml. ethyl alcohol;

Ultraviolet abs0rpzion.I-las an absorption peak at 250 to 254millimicrons at which the calculated specific extinction coefiicient isapproximately 5,000 to 7,000 based on a concentration of .00008 gram perml. of 95% ethyl alcohol;

Nitrogen. c0ntent.Approximately 2.4%

Sulfuric acid color test.When dissolved in concentrated sulfuric acid ona spot plate gives a reddish-brown color which changes to brown uponstanding.

When it is desired to eliminate some of the less desirable agents fromthe last described chloroform-soluble, partially water-soluble extract,the residual product obtained by the evaporation of the chloroform todryness is taken up in anhydrous ether and the ether extract evaporatedto dryness under vacuum.

More particularly, this residual chloroform-soluble product is extractedfive times with successive portions of anhydrous ether (ethyl ether),using 200 ml. of ether per 10 grams of material in each portion. Theether extract is then evaporated to dryness under vacuum. The residue isa light tan-colored resinous material containing desired hypotensivelyactive alkaloids free from some of the less desirable alkaloidscontained in the chloroform-soluble starting residue. The yield is about0.04% to 0.06%.

The light tan-colored product just described (Product E) is a desirabletherapeutic, blood-pressure-lowering alkaloidal fraction possessing thefollowing physico-chemical characteristics:

Solubility.Readily soluble in chloroform, dioxane, ether, benzene, ethylalcohol, methyl alcohol, propylene glycol, acetone and methylCellosolve; partially soluble in water;

Optical rotation range.[a] =-6 to -20 at a 11 concentration of 0.5 gramper 100 95% lethyhaleohol; I

Ultraviolet -absorption.-Has an absorption peak at approximately ,250millimicrons at which the calculated specific-extinction coetficient is4,500'to 7,500 based on a concentration of 0.00008 gram -per -ml. of 95%ethyl alcohol;

Nitrogen content-Approximately 2.3%;

. Sulfuric acid color test.When dissolved in concentrated sulfuric acidon a spot plate gives a reddish-brown color which-changes to brown uponstanding.

If desired, anhydrous benzol maybe substituted for the'anhydrous etherfor extracting the described :chlo'roform-soluble, partiallywater-soluble fraction, and .the product thereby obtained hasphysico-chemical characteristics which aresubstantially the same asthoseof the anhydrouscether extract.

Theabove described processes-may be used also for the recoveryaudvisolation of kindred alkaloids from similar alkaloid bearing plants.

,Havingparticular reference to ProductC of this invention, theconstituents are amorphous or noncrystalline forms 'of alkaloids whichare almost wholly water-insoluble. Apparently they are principallyunidentified amorphous bases possibly containing one or more of theknown amorphous alkaloids from Veratrum species.

However, .this composition is substantially free from cevadine (which iswater-soluble) and veratridine, these two together being sometimesreferred to as veratrine. Thisproduet is also substantially free fromthe commonly known Veratrum alkaloids particularly -jervine,rubijervine, isorubijervine, pseudojervine, veratramine, veratrosine,and the like. Thus, our process results in eliminating from the Veratrumalkaloids those alkaloids which are commonly crystalline and at the sametime produce therapeutically undesirable side reactions. In other words,we have been able to isolate an alkaloidal composition whcih containsonly the desirable hypotensively active alkaloids, only traces of theusual crystalline, objectionable alkaloids being found.

In-Product B, some ofthe crystalline, better known Veratrum alkaloidsare found, more particularly rubijervine and isorubijervine, but theproportion of these is small enough and the objectionablecharacteristics are so minor, at least in the presence of the otherconstituents, that Product B is highly desirable asa hypotensiveagentcxceptpossibly with highly sensitive patients.

While Product A contains larger proportions of the commonly knowncrystalline type of alkaloids, nevertheless, the high content ofamorphous constituents renders the composition much more desirable thanVeratrum products heretofore available, in that objectionable sidereactions are minor.

Products D and E are partially water-soluble,ainor phous alkaloidcompositions, with scarcely a trace of crystalline alkaloids, and havebeen "found to havevery good hypotensive characteristics, althoughapparently not the equivalent of those 'ofProduct C.

Wherethe term crystalline or crystallineetype is applied to variousalkaloids, it signifies those which are commonly'known as such and arerecoverable in crystalline form, and the term amorphous applies vtothose alkaloids which are now characteristically separable in amorphous'form. V

Inasmuch as many variations of the generic, invention herein "disclosed'willbecome apparent to 'those skilled in this art, it 'is intended toprotect all modifications falling withinthe scope of the patent claims.

We claim as our invention:

1. A-processfor isolating hypotensive alkaloids substantially-free-fromthose having objectionable side reactions,--said process including thesteps of: obtaining-fa solution of alkaloids from plants of the Veratrumfamily in arnmoniacal organic'solvent for said alkaloids; dissolving inan aqueous solution of a non-oxidizing, nonreducing a i t os a ka o uble therein which are found in ;said ammoniacal organic solvent;:addingammonium sulfate to such acid solution toprecipitatealkaloidshaving-therapeutically objectionable side reactions; rejecting ;theinsolubles formed in such acid solution; precipitating alkaloidfractionsfrom the resultant acid solution, following removal of said insolubles,by alkalizing with an agent from the class consisting of ammonia andalkali metal hydroxides; dissolving the last-mentioned precipitate inchloroform; evaporating to dryness; taking up from the residue inbenzene those fractions soluble therein; and recovering the benzenesoluble fractionsas a hypotensive composition of alkaloids.

'2. A process for isolating a mixture of hy'potensive alkaloids fromVeratrum plants containing the same, which process comprises the stepsof; obtaining a solution of the Veratrum alkaloids in an aqueoussolutionof a non-oxidizing, non-reducing acid; adding to said solution sulfateions'to precipitate sulfates of undesired alkaloids; rejecting theinsolubles formed thereby; rendering the-resulting solution alkaline toproduce a precipitate of alkaloids; dissolving thelast-mentionedprecipitate in chloroform; evaporating to dryness; takingup fromthe residue in benzene, those alkaloids soluble therein; andrecovering the benzene-soluble alkaloids as a hypotensive composition.

3. A process as defined'in claim 2, in which the solution of Veratrumalkaloids in said aqueous solution of said acid is obtainedby a seriesof steps including: extracting Veratrum plants withan organic alkaloidsolvent made alkaline with ammonia; replacing such solvent and ammoniawith a water-miscible organic alkaloid solvent; mixing the resultingsolutionwith an aqueous solution 'Of a non-oxidizing, non-reducing acid;and rejecting materials insoluble in said last-mentioned aqueoussolution.

4. ,A rprocess as defined in claim 3, in which the firstmentionedorganic alkaloid solvent is chloroform containing ammonia gas dissolvedtherein.

5. A process as defined in claim 3, in which the firstmentioned organicalkaloid solvent is chloroform containing ammonia gas dissolved therein;and said water-miscible organic alkaloid solvent is dioxane.

'6. A, process;as defined in claim 2, in which the solution of Veratrumalkaloids in said aqueous solution of said acid is obtained by a seriesof steps including: extracting Veratrum plants with chloroformcontaining ammonia gas "dissolved therein; replacing said chloroform andammonia with dioxane; mixing the resulting solution with an aqueoussolution of acetic acid;:and rejecting materials insoluble in saidaqueous solution of acetic acid.

.7. The process as defined in claim .6, in which the alkaloids solublein said aqueous solution of acetic acid are precipitated by renderingsaid aqueous solution alkaline; and-the precipitated alkaloids areseparated and again dissolved inanaqueous solution ofacetic acid.

58. A processas defined inclaim 2, in which the solu tionof thevVeratrum alkaloids in-said aqueous-solution of said acidis obtained bya series of steps including: extracting Veratrum plants with anaqueoussolution of acetic acid; rendering the extract alkalineto-precipitate Veratrum alkaloids; recovering in solution in chloroformthe chloroform-soluble alkaloids, the insolubles being rejec-ted;evaporating the resulting solution -ofalkaloids to dryness;'anddissolving the residue in an aqueous solution of a non-oxidizing,non-reducing acid.

9. A process as defined in claim 2, in which saidnonoxidizing,-non-reducing acid is acetic acid.

10. *Aprocess as defined in claim 2, in which the sulfate ions-are addedin the form of ammonium sulfate.

--1l. A process as defined in claim '2, in which the lastrncntionedcomposition is dissolved in acetone and any insoluble material andcrystalline precipitate forming therein is discarded; and the solublematerial recovered as a further purified hypotensive composition.

12. A process for isolating a mixture of hypotensive alkaloids fromVeratrum plants containing the same, which process comprises the stepsof: initially extracting the plants with chloroform containing ammoniagas absorbed therein to obtain a solution of alkaloids; replacing thechloroform and ammonia in said solution with dioxane; thereafterreplacing said dioxane with an aqueous solution of acetic acid whilerejecting materials insoluble in said acid solution; adding ammoniumsulfate to said acid solution to precipitate sulfates of undesiredalkaloids; rejecting the insolubles formed thereby; rendering theresulting solution alkaline with ammonia to produce a precipitate ofalkaloids; dissolving the last-mentioned precipitate in chloroform;evaporating the resulting chloroform solution of alkaloids to dryness;taking up the residue in benzene; discarding insoluble material;evaporating the remaining benzene solution of alkaloids to dryness;taking up the resulting residue in acetone; discarding any insolublematerial and any crystalline precipitate which forms in said acetone;and recovering the remaining acetone-soluble alkaloids as a hypotensivecomposition.

13. A process for isolating a mixture of hypotensive alkaloids fromVeratrum plants containing the same, which process comprises the stepsof: initially extracting the plants with chloroform containing ammoniagas absorbed therein to obtain a solution of alkaloids; replacing thechloroform and ammonia in said solution with dioxane; thereafterreplacing said dioxane with an aqueous solution of acetic acid whilerejecting materials insoluble in said acid solution; rendering said acidsolution alkaline with ammonia; filtering out the resultant precipitate;dissolving such precipitate in acetic acid; adding ammonium sulfate tosaid acid solution to precipitate sulfates of undesired alkaloids;rejecting the insolubles formed thereby; rendering the resultingsolution alkaline with ammonia to produce a precipitate of alkaloids;dissolving the lastmentioned precipitate in chloroform; evaporating theresulting chloroform solution of alkaloids to dryness; taking up theresidue in benzene; discarding insoluble material; evaporating theremaining benzene solution of alkaloids to dryness; taking up theresulting residue in acetone; discarding any insoluble material and anycrystalline precipitate which forms in said acetone; and recovering theremaining acetone-soluble alkaloids as a hypotensive composition.

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1. A PROCESS FOR ISOLATING HYPOTENSIVE ALKALOIDS SUBSTANTIALLY FREE FROMTHOSE HAVING OBJECTIONABLE SIDE REACTIONS, SAID PROCESS INCLUDING THESTEPS OF: OBTAINING A SOLUTION OF ALKALOIDS FROM PLANTS OF THE VERATRUMFAMILY IN AN AMMONIACAL ORGANIC SOLVENT FOR SAID ALKALOIDS; DISSOLVINGIN AN AQUEOUS SOLUTION OF A NON-OXIDIZING, NONREDUCING ACID THOSEALKALOIDS SOLUBLE THEREIN WHICH ARE FOUND IN SAID AMMONIACAL ORGANICSOLVENT; ADDING AMMONIUM SULFATE TO SUCH ACID SOLUTION TO PRECIPITATEALKALOIDS HAVING THERAPEUTICALLY OBJECTIONABLE SIDE REACTIONS; REJECTINGTHE INSOLUBLES FORMED IN SUCH ACID SOLUTION; PRECIPITATING ALKALOIDFRACTIONS FROM THE RESULTANT ACID SOLUTION, FOLLOWING REMOVAL OF SAIDINSOLUBLES, BY ALKALIZING WITH AN AGENT FROM THE CLASS CONSISTING OFAMMONIA AND ALKALI METAL HYDROXIDES; DISSOLVING THE LAST-MENTIONEDPRECIPITATE IN CHLOROFORM; EVAPORATING TO DRYNESS; TAKING UP FROM THERESIDUE IN BENZENE THOSE FRACTIONS SOLUBLE THEREIN; AND RECOVERING THEBENZENE SOLUBLE FRACTIONS AS A HYPOTENSIVE COMPOSITION OF ALKALOIDS.